FIG. 3.
Decreased processivity of RT containing the didanosine-selected Leu74Val mutation. Various amounts of WT and mutant RT lysates were assayed for RT activity with the poly(rA)-oligo(dT) system (see Materials and Methods), and the reaction products were analyzed on a 6.0% polyacrylamide-7.1 M urea sequencing gel. Reaction products with approximately equivalent amounts of RT activity are shown on a representative gel (A). M, size markers from a dideoxy sequencing reaction with a pBluescript plasmid and M13/pUC reverse sequencing primer (T-track only). RT activities for WT, Leu74Val, and Met184Val viruses were 3.0 × 104, 3.1 × 104, and 3.5 × 104 cpm of α-32P, respectively. Nucleotide lengths of cDNA synthesized for WT, Leu74Val, and Met184Val are 43, 33, and 33, respectively, in this assay. Numbers on the left indicate the nucleotide lengths. (B) Size distribution and relative amounts of cDNA synthesized by WT and mutant RTs. The median values for relative cDNA density from five independent assays were generated by using a statistical program and plotted with respect to sizes of cDNA (Table 2). Groups of five cDNA bands starting at the bottom of gel in individual lanes were quantified at one time, and the intensity of the entire lane was determined with ImageQuant (Molecular Dynamics) software. The bar diagram shows the relative amounts of cDNA synthesized by WT and mutant enzymes.